The present invention provides a method for isolation and assay of molecules of interest using affinity matrices in the presence of detergent. These isolation methods have application in various fields of biology such as protein purification, nucleic acid purification, high throughput assays, diagnostic assays, functional genomics, functional proteomics, phage display, and protein expression profiling.
Affinity separation techniques are increasingly used for the isolation or quantification of biological molecules. Chromatographic techniques that use magnetically attractable affinity particles or beads for separation of specific molecules from a liquid are well documented in biochemical, biomedical, and molecular biological research. Affinity separation techniques involve the suspension of finely divided affinity matrix particles in a solution that contains molecules of interest in an impure or dilute form. The molecules of interest are captured or immobilized on the matrix particles by virtue of specific or non-specific interactions between the molecules and an affinity ligand for such molecules associated with the surface of the affinity particles. The affinity particles and the bound target molecules of interest may then be separated or collected together using a variety of standard techniques, such as filtration, centrifugation, decanting, and the like.
Particularly useful materials and techniques, especially for automated separation processes, utilize magnetically attractable affinity beads. After binding the target molecule, application of a magnetic field to the vessel containing the magnetic affinity particles (beads) will cause them to migrate towards the source of the field, thus collecting and concentrating the beads at the wall of the vessel. With the magnetic field still applied, the remainder of the solution and unbound components (the supernatant) can be removed by pouring it off or by using a pipetting device, leaving the magnetically collected pellet of magnetic particles intact. Additional solution(s) can then be added and the magnetic field removed, thus allowing the beads to be resuspended. If the interaction between the magnetic affinity beads and the molecules of interest is disrupted (target molecule eluted), the molecules can be recovered by reapplying the field and removing and retaining the supernatant containing the purified/concentrated molecules of interest. If the affinity beads are being used in an assay, e.g., where only the presence of the target molecule needs to be determined or quantified, elution from the affinity matrix is not necessary and the beads having bound molecules of interest may be exposed to a detection reagent, such as a labeled antibody, enzyme, or the like.
Manual handling of affinity beads limits the possible throughput, and to increase throughput several automated and semi-automated technologies have been devised, for both single-sample and multi-sample vessels. Magnetic affinity beads, as mentioned above, lend themselves readily to such automated and semi-automated technologies, and practitioners are able to achieve a higher throughput by better separation of the magnetic beads and by increasing the number of samples processed per run. Processing can be increased by either moving the magnetic source in relation to the vessels (see, WO 96/31781) or by moving the vessel in relation to a single or multiple magnetic sources (see, U.S. Pat. Nos. 5,128,103 and 5,147,529). The throughput in certain instances has been increased by placing the magnetic source adjacent to the pipette tips containing the magnetic beads (see, WO 97/44671, WO 97/31105, and EP 0 691 541), or removing the liquid from the magnetic beads using an automated pipetting system (see, U.S. Pat. No. 5,458,785).
Many suitable affinity ligands are known for use in preparing affinity matrix particles capable of binding a molecule of interest. One class of ligands that has been used successfully in affinity separations is metal chelate linkers. Porath et al. introduced the use of metal chelate affinity chromatography for protein isolation in 1975 (see, Porath et al., Nature, 258:598-599 (1975)). This technology has since been used successfully in many types of separations (see, review articles, e.g., Lonnerdal et al., J. Appl. Biochem., 4:203-208 (1982) and Sulkowski, Trends in Biotechnology, 3:1-7 (1985)). Metal chelate affinity separation is based on the discovery that metal ions, such as nickel, copper, and zinc, bound to or immobilized on a solid substrate or matrix, such as agarose or silica gels, can take part in a reversible interaction with electron donor groups situated on the surface of proteins, especially the imidazole side chains of histidine. At a pH value at which the electron donor group is present at least partially in non-protonized form, the protein is bound to the chromatography gel and can subsequently be eluted by means of a buffer with a lower pH value, at which the electron donor group is protonized. Nitrilotriacetic acid (NTA), bound to the carrier matrix via a spacer, has been very reliable as the chelate donor (see, e.g., U.S. Pat. No. 5,047,513).
Regardless of the type of ligand used to capture the molecule of interest, affinity separation techniques suffer from a common problem of bead loss during the separation process. This problem has been observed during automated as well as manual bead handling each time the affinity beads are washed, molecules are eluted from the beads, or supernatant liquids are removed or retrieved. If the beads are collected at the bottom of a vessel, such as a microtiter plate, there is a high risk that while removing solution, some beads will also be removed. The loss or removal of beads leads to irreproducible results, inaccurate quantitation of bound materials, lower yields in purification protocols, and a lower throughput in assays.
Unexpectedly, it has now been shown that the use of small amounts of detergents in conjunction with use of the affinity beads, the loss of beads can be significantly reduced. The invention described herein makes possible the manual and automatic processing of affinity beads, especially magnetic beads, in multi-well and single well vessels. Commercially available multi-well and/or single well formats (e.g., microtiter plates) and robots (e.g., BioRobot) may be used.
The present invention provides separation methods that are highly flexible and which are characterized by high sample throughput without risk of sample mix-up. These and other aspects and advantages of the invention will be apparent from the description and examples presented below.
The invention provides methods for separating a finely divided particulate matrix from a solution so as to minimize loss of particles or beads from the matrix. More particularly, the present invention provides methods of isolating molecules of interest from a sample or solution using affinity particles or beads. The methods described herein provide the means for reducing loss of affinity particles or beads during a separation process, for increasing yields of target molecules of interest, and for increasing the accuracy and reproducibility of assay methods based on affinity separations.
In its broadest aspects, the present invention relates to a method of separating particles from a solution comprising the steps:
(a) combining a solution with a finely divided particulate matrix, in the presence of detergent;
(b) collecting the particles of the particulate matrix, e.g., by centrifugation, filtration, magnetic force (if the particles are magnetically attractable), etc.;
(c) removing supernatant solution.
Where the particles are affinity particles, the present invention provides a method for isolating a molecule of interest from a solution in a vessel, comprising the steps of:
(a) contacting the solution with affinity particles insoluble in the solution and capable of binding with said molecule of interest, in the presence of detergent; and
(b) separating the affinity particles from the rest of the solution, e.g., by introducing a magnetic field across the vessel (for magnetic affinity beads), centrifugation, filtration, etc.
Alternatively, the affinity particles may be treated with detergent prior to the contacting step.
The affinity particles and bound molecules of interest may be subjected to additional steps, depending on the object of the separation (i.e., whether the molecule of interest is being assayed, detected, recovered, or purified from the solution). Such additional steps include washing steps, e.g., in which the separated particles are resuspended in another solution; elution steps, in which the separated particles are subjected to solution conditions causing disruption of the affinity interaction between the particles and the molecules of interest, causing the molecules to be released from the particles; assay or detection steps, wherein the separated particles are contacted with detection reagents, such as labeled monoclonal antibodies specific for the bound molecule of interest.
In a particular embodiment of the invention, a magnetic separation method comprises:
(a) contacting a solution with magnetic particles in a vessel, in the presence of detergent;
(b) bringing the vessel and a magnet into proximity with each other, whereby said magnetic particles are immobilized within the vessel (i.e., by magnetic force);
(c) removing supernatant from the vessel;
(d) adding new solution to the vessel; and
(e) separating the vessel and the magnet and re-suspending the particles in the new solution.
In the foregoing embodiment, it will be recognized that steps (d) and (e) may be reversed, to the same effect.
The methods according to the present invention may be applied to the separation of a variety of molecules from various solutions. For example, molecules of interest may be peptides, polypeptides, proteins, nucleotides, nucleic acids, carbohydrates, lipids, complexes or organic molecules. Preferably, the methods of the present invention are used to isolate proteins and nucleic acids. More preferably, the methods are used to isolate 6x-His-tagged proteins.
Magnetic particles useful in preferred methods of the present invention may be any of a variety of materials including, for example, ferromagnetic beads, superparamagnetic beads, and combinations thereof. The particulate matrix materials that are advantageously separated according to the methods of the present invention may be any solid substrate materials insoluble in the solution(s) in which they are suspended. Typical examples include particulate agarose, silica, nitrocellulose, cellulose, acrylamide, latex, polystyrene, polyacrylate, polymethacrylate, polyethylene polymers such as polyvinyl alcohol, glass particles, silicates such as calcium, magnesium and aluminum silicates, metal oxides such as titanium oxides, tin oxides, etc., apatites, and the like, and combinations thereof.
The detergents used in the methods of the present invention are nonionic, anionic, zwitterionic, and cationic detergents, and combinations thereof. Preferably, the concentration of detergent used in the methods of the invention is at least about 0.0005-2.0% (v/v). In a preferred embodiment, the detergent is a nonionic detergent selected from a group consisting of Tween 20, Triton X-100, Nonidet P-40, and combinations thereof. In another embodiment of the invention, the detergent is the cationic detergent dodecyltrimethyl ammonium chloride. In another embodiment of the present invention, the detergent is an anionic detergent selected from a group consisting of sodium dodecyl sulfate (SDS), sarkosyl, and combinations thereof. In another embodiment of the invention, the detergent is the zwitterionic detergent CHAPS.
According to the methods of the present invention, the concentration of detergent should not exceed about 2% (v/v). Preferably, the concentration of a nonionic detergent according to the methods of the present invention is at least about 0.005% (v/v). Preferably, the concentration of an anionic detergent according to the methods of the present invention is at least about 0.05% (v/v) and does not exceed about 1% (v/v). Preferably, the concentration of a cationic detergent according to the methods of the present invention is at least about 0.5% (v/v) and does not exceed about 1% (v/v). Preferably, the concentration of a zwitterionic detergent according to the methods of the present invention is at least about 0.01% (v/v) and does not exceed about 2% (v/v).
Most preferably, the nonionic detergent Tween 20 is used at a concentration of at least about 0.05% (v/v) in the methods of the present invention when used in the isolation of nucleic acids or proteins.
This invention provides, in an affinity system, an improvement in signal, signal to noise ratio, reproducibility, and yield, by adding a small amount of detergent to the solution or treating the matrix particles or beads with a small amount of detergent.